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Acquired thrombotic thrombocytopenic purpura (TTP) is characterized by the presence of neutralizing and/or non-neutralizing anti-ADAMTS13 autoantibodies which are predominantly of the IgG class and less frequently of the IgM and/or IgA classes [1–4]. However, the extent to which these antibodies circulate in plasma in their free form or as a complex with ADAMTS13 and how this proportion changes over time is unclear. We and others have recently reported indirect detection by an ADAMTS13 antigen (ADAMTS13:Ag) ELISA of IgG ADAMTS13-specific immune complexes (ICs) in plasma from patients with acquired TTP [5,6]. In the present study we used another more direct method for detecting ICs and compared the levels of free anti-ADAMTS13 antibodies, ADAMTS13:Ag, ADAMTS13 activity (ADAMTS13:Ac) and inhibitor with those of ICs.

Samples from a patient with acquired idiopathic TTP, whose case history has in part been described previously [1], were analyzed. Treatment modalities of this patient are summarized in Fig. 1A. The patient presented with undetectable ADAMTS13:Ac and ADAMTS13:Ag, but also without ADAMTS13:Ac inhibition as determined by the CBA assay [1,5,7]. Re-assessment of the inhibitor with the more sensitive fluorometric FRETS-VWF73 assay [7] revealed low level inhibition of ADAMTS13:Ac, corresponding to approximately 0.8 Bethesda units (BU) mL−1.

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Figure 1.  Course of ADAMTS13-related variables in a patient with acquired idiopathic thrombotic thrombocytopenic purpura (TTP). (A) Treatment regimen over 64 days until the patient’s death. IAS, immunoadsorption (on staphylococcal Protein A Sepharose columns); PEX, plasma exchange (40 mL fresh-frozen plasma per kg body weight); CS, corticosteroids (prednisone 100, 50, 25, 12.5 mg per day). The date of the splenectomy is also indicated. The following ADAMTS13-related variables were determined at the time points indicated: (B) ADAMTS13:Ac (FRETS-VWF73, U mL−1), ADAMTS13:Ag (ng mL−1, reference range 740–1420 ng mL−1) and ADAMTS13 inhibitor (BU mL−1); (C) IgG anti-ADAMTS13 antibodies (titer) and IgG-immune complexes (ICs) (arbitrary units), (D) IgA anti-ADAMTS13 antibodies (titer) and IgA-ICs (arbitrary units); (E) IgM anti-ADAMTS13 antibodies (titer) and IgM-ICs (arbitrary units). ADAMTS13-specific ICs were analyzed by co-immunoprecipitation of ADAMTS13 with either Protein G Sepharose or anti-IgA- or anti-IgM-specific affinity matrixes. Plasma-bound antibodies were eluted and co-immunoprecipitated ADAMTS13 was analyzed by Western blot using an affinity-purified polyclonal rabbit IgG anti-human ADAMTS13 antibody. The relative amount of ICs (expressed in arbitrary units) was quantified by densitometry analysis and rADAMTS13 (A; 1 ng) as standard. Two pooled normal human plasmas (N1 and N2) were used as a negative control. The insets in C–E show a section of the respective blots. The entire blots and a detailed description of the method are depicted in Fig. S1.

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Figure 1B summarizes the dynamic course of ADAMTS13:Ac, ADAMTS13:Ag and ADAMTS13 inhibitor during the 64 days’ follow-up until the patient’s death. Analyzes of specific IgG, IgM and IgA anti-ADAMTS13 antibodies by ELISA [8] detected IgG and IgA antibodies at most time points, whereas IgM antibodies were only measurable until day 12 (Figs 1C–E and S1). The patient’s anti-ADAMTS13 IgG subclass profile on admission (82% IgG1, 7% IgG2, 11% IgG3, no IgG4) did not change significantly during the course of the disease (not shown). The IgG and IgA antibody titers decreased during periods of transient response to plasma exchange (PEX) treatment, but this drop was not always accompanied by a recovery in ADAMTS13:Ac and ADAMTS13:Ag levels. This lack of ADAMTS13 activity recovery and the presence of high levels of IgG1 combined with undetectable IgG4 have been described as indicators of a poor prognosis and an increased risk of mortality in patients with acquired TTP [6,8] and may, at least in part, explain the poor outcome of the patient.

The presence of ICs was demonstrated by co-immunoprecipitation of ADAMTS13 with immunoglobulins, using protein G for the isolation of total IgG, and anti-IgA- and anti-IgM-specific affinity matrixes for isolation of total IgA and IgM, respectively (Fig. 1C–E). Interestingly, ADAMTS13-specific IgG-ICs were detectable in significant amounts at most times during the 2-month follow-up, particularly when the free IgG antibody titers and inhibitor were low. By contrast, IgG-ICs became almost undetectable when the free IgG antibody titers and ADAMTS13 inhibitor were high (Fig. 1B,C). Thus, IgG-ICs followed an inverse kinetics to both the free IgG antibody in plasma and the inhibitory titer. Similar trends were seen for the IgA- and IgM-specific ICs, but with an apparently lower total concentration than IgG-ICs (Fig. 1D, E). Notably, low amounts of IgM-ICs were detected at most time points although no free IgM antibodies were detected after day 12 (Fig. 1E), suggesting that all free IgM antibodies had been complexed by ADAMTS13. Overall it appears that anti-ADAMTS13 antibodies are not detected by ELISA when they are part of an immune complex and the inhibitor titer measured correlates with the amount of free anti-ADAMTS13 antibodies.

In the patient studied, samples from most time points were devoid of free ADAMTS13:Ag but contained circulating ICs, indicating that ADAMTS13, either newly synthesized or exogenously administered by plasma infusion during PEX, had been quantitatively sequestered through formation of ICs. ADAMTS13:Ag was also undetectable in samples that had high levels of free anti-ADAMTS13 antibodies but lacked circulating ICs (Fig. 1; days 8–11 and 16–21), suggesting that ICs that had formed were already cleared at these time points.

Considering that the patient was receiving daily PEX, the plasma concentration of antibodies and antigen changed continuously throughout therapy, thereby influencing the dynamic processes of formation, biological activity, deposition and clearance of ICs. Under normal conditions, ICs are thought to be quickly cleared by the reticulo-endothelial system, and only a continuous formation eventually exceeds the clearance capacity of the system, causing a build-up of ICs in the circulation. There, IgM- or IgG-containing ICs can either activate the complement classic pathway or bind to cellular Fc receptors [9,10]. Circulating IgA-ICs have the potential to activate complement via the mannan-binding lectin pathway and immune cells when binding to Fcα receptors. These features can lead to endothelial cell activation, promoting inflammation and disturbing the hemodynamic flow, which in turn may contribute to enhanced VWF-dependent platelet adhesion and aggregation. The continuous presence of excessive amounts of circulating ICs might therefore perpetuate a pro-inflammatory state promoting thrombosis and predisposing to a relapse.

In conclusion, the present study on the dynamic course of the ADAMTS13-specific autoantibody response in a patient with refractory TTP suggests that in addition to a characterization of the whole panel of autoantibodies against ADAMTS13, circulating ADAMTS13-specific ICs also need to be determined to obtain a full picture of the status of the disease. Formation of ADAMTS13-specific ICs capturing all freely available ADAMTS13 should be particularly considered in patients who are refractory to treatment. Future studies on a representative cohort of patients with acquired TTP will show whether our observation of an inverse correlation of free and IC-sequestered anti-ADAMTS13 antibodies in a single patient is a general phenomenon.

Disclosure of Conflict of interests

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  2. Disclosure of Conflict of interests
  3. References
  4. Supporting Information

S. Ferrari, B. Plaimauer, P.L. Turecek, K. Varadi, H. Rottensteiner and F. Scheiflinger are full-time employees of Baxter Innovations GmbH, Vienna, Austria. The other authors state that they have no conflict of interest.

References

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  4. Supporting Information
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Supporting Information

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  2. Disclosure of Conflict of interests
  3. References
  4. Supporting Information

Figure S1. Detection of circulating ADAMTS13-specific ICs at different time points by co-immunoprecipitation and Western blotting.

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JTH_4548_sm_FigS1.pdf334KSupporting info item

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